The osteochondral (OC) tissues of hyaline articular cartilage, calcified cartilage and subchondral bone operate collectively, thus the degeneration of any one inevitably influences the others. Pressurization of interstitial fluid in healthy cartilage protects cells from excessive tissue strain under large joint contact forces. In osteoarthritis (OA), extracellular matrix degradation results in vastly increased cartilage tissue permeability and diminished mechanical properties and function. Concomitantly, degenerative processes (e.g., vascular invasion and microfractures) create new routes for fluid efflux through calcified cartilage and into the underlying subchondral bone. Thus mechanical and biochemical signaling between OC tissues are perturbed. Moreover an interplay exists between tissue strain and interstitial fluid flows (iFFs) that stimulates bone cells to alter bone structure and, consequently, their mechanical environment (e.g., subchondral bone thickens substantially in late-stage OA). In particular, osteocytes, which are resident cells within bone, sense and respond to iFF changes, to disrupt homeostatic regulation of bone mass through several established mechanosensory pathways (e.g., PGE2 and Wnt/?-catenin signaling). Thus our overarching hypothesis for this research is that the changes to cartilage at the onset of OA, which lead to higher permeability, affect the iFF in subchondral bone and alter osteocyte signaling by releasing PGE2 among other molecules and activating Wnt/?-catenin signaling, which signals osteoblasts and leads to the observed pathophysiological phenotype of increased subchondral bone mass. To test this hypothesis, we will establish a novel 3D tri-layered hydrogel that emulates the complex flow behavior of OC tissues under dynamic compressive forces. Tri-layered hydrogels will be designed with a soft layer that experiences large strains and induces iFF into the bony layer, an intermediate layer whose crosslink density is tuned for permeability to control fluid velocity, and finally a stiff bony layer comprised of an engineered lacunocanalicular network of osteocytes that experience highly dynamic fluid flow with minimal strains. We have outlined two specific aims. In specific aim #1, we will develop tri-layer hydrogels that possess moduli and permeability characteristics to emulate iFF behavior of healthy and osteoarthritic bone tissue using a combined experimental and computational approach. In specific aim #2, we will perform a series of experiments to study the mechanisms by which osteocytes, when embedded in an engineered lacunocanalicular network in the stiff bony layer of the tri-layer hydrogels, respond to different levels of iFF. Upon completion of this project, we expect to have developed a tri-layered hydrogel that captures the unique iFF behavior in healthy and osteoarthritic OC tissues and determined the role of iFF in mediating osteocyte signaling. Long-term, this platform will enable us to study the dynamic conversation between chondrocytes, osteoblasts, and osteocytes under healthy and osteoarthritic mechanical environments and provide novel insights into the role of mechanical cues in the progression of whole joint OA. !